**2. Overview**

In view of the above mentioned and widely accepted explanations and morphological descriptions of crystal, nuclei formation and phase transformations, the focus of this contribution relies on recent advances in the physical understanding of the disorder-order, fluid-crystal-fluid transitions of Lipid A-phosphates, the formation of a re-entrant phase, particularly for Lipid A-diphosphate e.g freezing and melting, and the molecular mimicry of hierarchical self-assembly of Lipid A-phosphates. This was achieved using a variety of experimental techniques e.g. scanning electron microscopy (SEM), high resolution transmission electron microscopy (HRTEM) and small-area electron diffraction (SAED), small-angle X-ray diffraction & solution scattering (SAXD & SAXS), static & quasi-elastic light scattering (LS & QELS). The contribution is outlined as follows: In remainder of the brief introduction, a short description is given which is devoted to the chemistry and importance of this class of molecules ("nano-medicine") in the day-to-day life. In Section 3, the phase transition of Lipid A-diphosphate clusters, the formation of various crystal forms as a function of particle number density (*n*) or volume fraction (), ionic strength (*I*) and T will be introduced since they affect the crystallization in 2d and 3d. Observations on freezing and melting of Lipid A-diphosphate clusters on a physical molecular level are also presented. The occurrence of the re-entrant fluid phase of Lipid A-diphosphate clusters upon addition of M NaOH after a crystalline BCC phase will be compared with the various crystalline phases upon decreasing *I* and increase of *n* for NaCl. The self-assembly of Lipid A-diphosphates clusters composed of different "subunits" e.g. six-hexaacylated chains and the corresponding Lipid A-diphosphate with four acylated fatty acid chains with the same phosphorylated disaccharide will be discussed in more detail (crystals, symmetry packing). These crystalline Lipid A-diphosphate cluster complexes may be called "*autovaccines*" (*Note:* An immunizing agent is composed of a selected or modified chemical entity of an original microorganism or virus, which does not cause clinical signs associated with the parent microorganism or virus, but still infects & multiplies in the host so as to induce immunity). Section 4 reports on the formation of Lipid A-diphosphate crystals by surface-induced gradient-induced crystallization in monolayers, their thermodynamics with relation to surface tension and their morphologies. In Section 5 the conclusions are presented.

#### **2.1. Chemistry and the biological role of Lipid A-diphosphate**

The lipopolysaccharides (*LPS)* are a group of diverse lipid-containing carbohydrates that exhibit a wide variety of biological activities. They occur naturally on the outer cell membranes of Gram-negative bacteria such as *Escherichia coli.* Although the lipopolysaccharides are large molecules, most of their biological activities result from the activity of a small portion of the molecule known as Lipid A-diphosphate. The structure of Lipid A consists of two β (1,6)-linked D-glucosamine units with polar phosphate groups at 1 and 4' positions (Fig. 1) [28]. This problem caused approximately 21 000 mortalities in 1996 in the U.S. alone [29].

98 Recent Advances in Crystallography

**2. Overview** 

from self-assembly of colloidal materials [26, 27].

numerous applications, especially for colloidal particles [21-23], biomimetic approaches to mineralization [24], curved crystalline shapes that emerged from mixtures of barium or strontium carbonates and silica in alkaline media [25], and usually from devices derived

In view of the above mentioned and widely accepted explanations and morphological descriptions of crystal, nuclei formation and phase transformations, the focus of this contribution relies on recent advances in the physical understanding of the disorder-order, fluid-crystal-fluid transitions of Lipid A-phosphates, the formation of a re-entrant phase, particularly for Lipid A-diphosphate e.g freezing and melting, and the molecular mimicry of hierarchical self-assembly of Lipid A-phosphates. This was achieved using a variety of experimental techniques e.g. scanning electron microscopy (SEM), high resolution transmission electron microscopy (HRTEM) and small-area electron diffraction (SAED), small-angle X-ray diffraction & solution scattering (SAXD & SAXS), static & quasi-elastic light scattering (LS & QELS). The contribution is outlined as follows: In remainder of the brief introduction, a short description is given which is devoted to the chemistry and importance of this class of molecules ("nano-medicine") in the day-to-day life. In Section 3, the phase transition of Lipid A-diphosphate clusters, the formation of various crystal forms as a function of particle number density (*n*) or volume fraction (), ionic strength (*I*) and T will be introduced since they affect the crystallization in 2d and 3d. Observations on freezing and melting of Lipid A-diphosphate clusters on a physical molecular level are also presented. The occurrence of the re-entrant fluid phase of Lipid A-diphosphate clusters upon addition of M NaOH after a crystalline BCC phase will be compared with the various crystalline phases upon decreasing *I* and increase of *n* for NaCl. The self-assembly of Lipid A-diphosphates clusters composed of different "subunits" e.g. six-hexaacylated chains and the corresponding Lipid A-diphosphate with four acylated fatty acid chains with the same phosphorylated disaccharide will be discussed in more detail (crystals, symmetry packing). These crystalline Lipid A-diphosphate cluster complexes may be called "*autovaccines*" (*Note:* An immunizing agent is composed of a selected or modified chemical entity of an original microorganism or virus, which does not cause clinical signs associated with the parent microorganism or virus, but still infects & multiplies in the host so as to induce immunity). Section 4 reports on the formation of Lipid A-diphosphate crystals by surface-induced gradient-induced crystallization in monolayers, their thermodynamics with relation to

surface tension and their morphologies. In Section 5 the conclusions are presented.

The lipopolysaccharides (*LPS)* are a group of diverse lipid-containing carbohydrates that exhibit a wide variety of biological activities. They occur naturally on the outer cell membranes of Gram-negative bacteria such as *Escherichia coli.* Although the lipopolysaccharides are large molecules, most of their biological activities result from the

**2.1. Chemistry and the biological role of Lipid A-diphosphate** 

**Figure 1.** Chemical structures of Lipid A-diphosphate (A) and two Lipid A-diphosphate derivatives (B & C) with strong biological implication on the anti-inflammatory mediator level [28, 30]. Lipid Adiphosphate from *E. coli* is a 1,4-di-phosphorylated β-1,6-linked D-glucosamine disaccharide with four residues of amide-and O-esterified R-(-)-3-hydroxy fatty acids (\* denotes the chiral centers in the hydroxy fatty-acid esters), apart from the chiral and epimeric carbons in the glucosamine moieties which are not marked. The antagonistic Lipid A-diphosphate molecules shown in B & C contain the same disaccharide as in (A); however, they differ in the number anchored carbohydrate positions and the number of chiral fatty-acid chains but the chain lengths is the same. The monophosphate of Lipid A is only phosphorylated at the reducing end of the disaccharide (C-1).

The Lipid A-diphosphate is associated with lethal endotoxicity, pyrogenicity and specific immune response. It is also responsible for triggering a cascade of cellular mediators, e.g. tumor necrosis factor , interleukins, leukotrienes, thromboxane A2 from monocytes and macrophages. The Lipid A-diphosphate and their analogues are distinct from normal lipids with respect to structure, chirality and chemical building units (Fig. 1) [30]. The 2 and 2' amino positions and the 3 and 3' hydroxyl groups are esterified with hydroxy fatty acids. It is known that natural Lipid A-phosphates and approximants are potent immunostimulants which induce a number of desirable effects but also some undesirable ones [30]. Various analogues of Lipid A-diphosphate have been developed to avoid such unwanted effects as toxicity and pyrogenicity, and therefore, they are very distinct from other lipids and surfactants.

Lipid A-diphosphate and approximants possess beneficial effects in clinical therapy against chronic inflammatory diseases and are capable of decreasing resistance to antibiotics and cationic antimicrobial peptides (CAMP) [31]. In this context the CAMP play also a significant role in the immune reaction to gut commensals (inflammatory bowel disease (Crohn), ulcerative colitis) and possibly in antibiotic resistance [30, 31]. This infers to an increased bacterial invasion of the surface of the respective tissues accompanied by the loss of the protective barrier. This accounts for bacterial contamination of the intestinal surface where host and invader are physically in close contact. Accordingly, this view strongly supports the production of "*intestinal autovaccines*" and its therapeutic potential e.g for protection of CAMP synthesis and sustaining remissions. The chemical structure of the proinflammatory component of *LPS,* Lipid A (Fig. 1), varies between bacteria of different species where the Gram-negative bacteria modulate the structure of their *LPS*.
